Method of diagnosis

ABSTRACT

A method of identifying the presence or absence of an infected state in the living body by obtaining a biological fluid sample from a living body, adding to the sample a material known to bind with immune complex, adding to samples of the resulting material labelled reagents known to react specifically with antigen portions of different immune complexes, separating excess of the reagents and detecting the presence or absence of the label in the residual mixtures thereby to identify the nature of any reacted antigen portion of immune complex therein.

This is a continuation of application Ser. No. 616,963, filed 6/4/84, asPCT GB81/00232, Oct. 23, 1981, published as WO82/01593, May 13, 1982,now abandoned, which is a continuation of United States patentapplication Ser. No. 395,078, filed June 11, 1982, by David Parratt andentitled, "A Method of Diagnosis, now U.S. Pat. No. 4,548,909.

This invention relates to a method of diagnosis.

When a patient is infected by a micro-organism, or antigen, the entry ofthe micro-organism into the patient's bloodstream causes the productionof an antibody by the patient's body and the antibody combines with anantigen from the micro-organism and with "complement", which iscontained in the blood, to form an immune complex in the patient's body.

Recently immune complexes have been implicated in the aetiology of somehuman diseases, in particular as a cause of tissue damage, and thus theaccurate measurement of complexes is of growing importance to medicalpractitioners. Many of the procedures currently used for theirmeasurement rely upon interaction of complex with the complement system,for instance by virtue of the decreases in serum complement levels socaused. Alternatively, complex may be detected by measuring differencesin their physico-chemical characteristics relative to uncombinedantibody. These techniques, however, are generally insensitive,inaccurate, difficult to use and give little information about theantigen and antibody components of the complex. Other techniques makeuse of living cells or human reagents and as such are unsuitable forapplication on the extensive scale required by hospital laboratories.

During the initial stages of infection the patient's body contains anexcess of infecting micro-organisms over antibody but as the infectionproceeds the body produces more antibody until there is an excess ofantibody. In the early stages only a small amount of complex is formedin view of the small amounts of antibody produced by the body, and thisdoes not activate the complement, so little damage is caused to the bodyand no symptons are detected. As the production of antibody increaseshowever the amount of complex increases correspondingly, and as thestage approaches where the antigen and antibody are in equal proportionsin the body the complex is present in substantial amounts. Moreover, ithas been found that the complex produced at this stage has a half-lifeof about a week, so its effect is felt over a prolonged period. Onpassing the antigen/antibody balance point the antibody is produced inexcess and it has been found that the half-life of the complex producedthen is reduced to a few hours. Thereafter the antibody excess increasesuntil the antigen and the complex are no longer present.

In general, symptoms appear in a patient only during the period ofantibody excess, and this may be due to the high concentration ofcomplex present, both of long half-life from before the antigen/antibodybalance point and of short half-life from during and after the balancepoint. By the time that the symptoms appear, therefore, the infection iswell advanced and treatment may have to be drastic. Further, thesymptoms often appear after the infectious stage has been passed so thatthe infection may have been already passed on to others beforedetection.

It has previously been proposed to determine the amount of immunecomplex in a sample by means of an assay using conglutinin, as thismaterial has the property of combining with an immune complex. The useof conglutinin has however been restricted to the assay.

According to the present invention there is provided a method ofdiagnosis comprising providing a material for attachment to an immunecomplex, obtaining an extract from a living body, treating the bodyextract with said material thereby to attach any immune complex presentin the extract to said material, detecting the presence of attachedimmune complex, treating the attached immune complex with a series ofdifferent reagents or reagent mixtures known to react with specificimmune complex components, and detecting the presence or absence ofreaction between each reagent or reagent mixture and the immune complexthereby to identify the immune complex.

By using the method of this invention it is possible to test individualpatients for the presence or absence of complex and to identify thecomplex, at an early stage of complex production in the body and beforethe formation of damaging long-half-life complex. This can be done incircumstances, for example, where an infection has been detected bysymptoms in an individual and it is desired to prevent the infectionspreading to others. Thus those individuals who have been in contactwith the infected patient can be tested by the present method beforesymptoms appear, and isolated and treated if the diagnosis shows thatthey have the infection.

In an alternative example, regular diagnosis by the present method canbe carried out in environments where infections could spread rapidly andwith great effect, for example in schools, old peoples' homes and otherplaces where large numbers of people come together regularly. In thisexample the individuals can be "screened" periodically to detect anddiagnose any infections before they would otherwise become apparent andbefore they have reached a stage where they can be transmitted toothers.

The present method also allows infections to be treated early and canreduce thereby the amount of suffering of a patient. As the infectionmay be detected and diagnosed before the damaging long-half-life complexis produced, antibody specific to the offending antigen can beadministered in excess so that the antigen/antibody balance point israpidly reached and passed, and only a small amount of complex having along half-life is produced. The patient therefore would experienceconsiderably less discomfort as the symptoms would be reduced inintensity.

The material for attachment to the complex is preferably conglutinin asthis is the most convenient material available at this time; however,other materials may be used if appropriate, for exampleimmunoconglutinin or rheumatoid factor. Immunoconglutinins are naturallyoccurring antibodies formed against complement components in anantigen/antibody/complement complex, and conveniently react specificallywith complement which has been altered by interaction with complex.Immunoconglutinins may be obtained from the sera of most animals, e.g.sheep or rabbits which have previously been challenged with suitablecomplexes.

The conglutinin may be prepared by treating conglutinin-containing serumwith an absorbent for conglutinin, washing the treated serum to separatethe absorbed conglutinin, and separating the conglutinin from theabsorbent.

Preferably, the absorbent is zymosan, which may be obtained by treatingbaker's yeast with 0.1M 2-mercaptoethanol for 2 hours and alkylating theproduct with 0.02M iodacetamide in phosphate-buffered saline solution.

Preferably also the conglutinin is separated from the absorbent bycentrifuging and dialysing. Purification can be by chromatography, forexample by passage through Sephadex G-200.

Conglutinin advantageously reacts only with a C_(3b) component ofcomplement which has been formed after interaction with anantigen/antibody complex. Conglutinin may be obtained from the sera ofruminant animals, preferably from a euglobulin fraction of bovine serum,although it may also be obtained from the serum of other ruminants e.g.African buffalo, water buck, Uganda kob etc. In a particularly preferredmethod, conglutinin is extracted from bovine serum by use of zymosan, ayeast cell wall preparation. Conglutinin may also be separated fromother serum constituents by gel filtration, for instance throughSephadex G-200 in the presence of mercaptoethanol and EDTA, as describedby Lachman (Advances in Immunology 1967, 6, 479). Conglutinin richpreparations may be obtained by the combination of these procedures inany sequence to achieve the standard of purity desired.

The material, especially when it is conglutinin, is preferably attachedto a solid substrate prior to treatment with the immune complex, and ithas been found to be very effective to use as the substrate balls ofplastics material such for example as those sold by Euro-Matic Limited,which offer an extensive surface area over which the material can attachitself. Alternatively the solid substrate can be in the form of areceptacle such as a dish or a test tube, or can be coated on thesurface of such a receptacle.

Conglutinin and immunoconglutinin can be linked to the solid substrateby any suitable means. The conglutinin and immunoconglutinin arepreferably in a purified state, though impure or partially purifiedforms may be used such as the complete euglobulin fraction of bovineserum. The method used to link the conglutinin or immunoconglutinin tothe solid substrate may include use of a coupling reagent and/orappropriate preparation of the solid substrate. For example it has beenfound that conglutinin may be linked to Sephadex particles which havebeen activated with cyanogen bromide.

In the step of identifying the immune complex component reagents can beused which are active against either the antigen or the antibody. Aneffective method is to prepare mixtures of known reagents and to testsamples of the immune complex against these mixtures. If a mixture isfound which reacts with the component of the complex, either furthermixtures or individual ingredients of that mixture can then be testeduntil the active ingredient is recognised, thus allowing identificationof the component of the complex. Reagents active against the antigencomponent, known as anti-antigens, are preferably employed.

The presence or absence of reaction can for example be detected bylabelling the reagent with a radioactive label, and after treatment ofthe immune complex testing for the presence of radioactivity. In analternative method the reagent may be enzyme labelled, and reactiondetected by adding an enzyme substrate and determining a colour change,for example by spectrophotometry.

Embodiments of the method of this invention will now be described by wayof illustration in the following Examples.

EXAMPLE 1

Preparation of Conglutinin

A conglutinin-rich bovine serum is selected by a red cell clumping test,as described on pages 172 to 175 of "The Serology of Conglutination andits Relation to Disease" by Coombs et al (Blackwell, 1961). 1 liter ofhigh titre bovine serum (1 in 640) is mixed with 150 g of zymosan for 2hours at 4° C. The zymosan product is then washed with Veronal bufferedsaline solution and eluted with 0.01M PBS-EDTA for 10 minutes at roomtemperature. 0.5 ml/ml of yeast suspension is used. The product is thencentrifuged and the supernatent liquid containing the conglutinin isdialysed against 0.5M NaCl overnight at 4° C.

The liquid then has its pH adjusted to 3.0 using N HCl and is dialysedagainst 0.001N HCl and 0.5M NaCl for 2 hours.

Pepsin is added to the resulting liquid in an amount of 0.5 mg per ml in0.01N HCl to a final concentration of 2%. The solution is allowed tostand at 4° C. for 18 hours, and a buffer solution of 0.1M Na₂ HPO₄ and0.1N NaOH is added until a pH of 7.2 is achieved. The solution is thendialysed against 0.02M PBS-EDTA and then passed through a Sephadex G-200chromatography column to separate out the conglutinin which isidentified by the red cell test.

Assay and Diagnosis

1/4" polystyrene balls obtained from Euro-Matic Limited, are incubatedin a solution of conglutinin in a carbonate buffer in an approximateconcentration of 100 μg/ml, at pH 9.6. The balls, with the conglutininon their surface, are then washed 3 times in veronal bufferedsaline/Tween solution (0.1%v/v), and placed in test tubes.

A standard of aggregated human IgG is prepared by aggregating IgG at 63°C. for 30 minutes, and a stock of solution at a concentration of 1000μg/ml is diluted in 350 μl volumes to a concentration of 1 μg/ml. Toeach dilution is added 50μ of fresh normal human serum, and the mixtureis incubated at 37° C. for 30 minutes to allow complement fixation. Thetotal 400 μl volumes of the standard and of the Test sample are added tothe coated polystyrene balls in separate test tubes and incubated for 3hours at room temperature. The balls in each tube are then washed 3times with the veronal buffered saline/Tween solution at roomtemperature. 0.4 ml of radiolabelled anti-human immunoglobulin is addedto each tube and incubated for 3 hours at room temperature, then beingwashed 3 times with the veronal buffered saline/Tween solution. Theradioactivity of the bound immunoglobulin in the standard and testsamples is then measured, the standard providing a datum for determiningthe presence of immune complex quantitatively in the test sample.

Having measured the level of immune complex, further identical testsamples are added to conglutinin-coated balls, incubated and washed asdescribed above. Thereafter different selected mixtures ofrdioactive-labelled anti-antigens, which are known antibodies againstbacteria, fungi or viruses, are added to the test samples, incubated andwashed as described above, and the coated balls are then tested forradioactivity using an ICN Gammaset 500 counter.

When a mixture of anti-antigens is found to have bound to the immunecomplex, the components of that mixture are tested individually or infurther mixtures against the immune complex in the same manner until theanti-antigen which has bound to the complex is identified.

In this way not only the quantity of immune complex present in thesample but also the nature of the antigen in the complex can bedetermined, thus providing a quantitative and qualitative test forimmune complex.

The invention as described in this Example therefore provides adiagnostic test for identifying infection in a test sample.

EXAMPLE 2

This Example shows that early detection and identification of immunecomplexes can be made, before symptoms are apparent. Routine samples ofblood serum was obtained from two individuals, both young, apparentlyhealthy, adult males. The samples were each subjected to assay as inExample 1, and then diagnosis was carried out by adding to theconglutinin-coated balls with the complex attached a series ofnon-radiolabelled anti-antigens, namely:

anti-influenza B viruses

anti-respiratory syncytial viruses

anti-adeno viruses and

anti-influenza A viruses

The balls were then treated with radiolabelled sheep anti-human IgG,polyvalent reagent, and then tested for radioactivity after washing. Theballs treated with anti-influenza B produced a positive result.

The antibodies used were prepared in vivo but it is equally effective touse monoclonal antibodies.

The day after sampling, both individuals developed flu-like symptomswhich became worse throughout that day and into the next. By the thirdday they were both improving and on the fourth were able to return towork where blood samples were taken. Further blood samples were obtained16 days later and conventional serological analysis of these (i.e. byantibody detection) confirmed that both had been infected with a strainof Influenza B virus.

The results obtained with an immune complex assay are summarised inTables 1 and 2. The assays were carried out as described in theExample 1. Plastic balls were used to carry the conglutinin,radiolabelled sheep anti-human IgG identified the immune complexes andquantitated them, and radiolabelled rabbit anti-influenza B antibody wasthe anti-antigen which identified the antigen.

                  TABLE 1                                                         ______________________________________                                        Results of conglutinin radioimmunoassay for                                   immune complex                                                                            Corrected count                                                               Radioactivity in                                                                          μg/ml equivalents of                               Sample      cpm         immune complexes                                      ______________________________________                                        Standard                                                                      (1)         23010       600                                                   (2)         13590       300                                                   (3)         8690        150                                                   (4)         3750         75                                                   (5)         3670         37                                                   First Patient                                                                 Day 1       15450*       350*                                                 Day 4       8710        190                                                   Day 20       480        <75                                                   Second Patient                                                                Day 1       22460*       540*                                                 Day 4       7900        170                                                   Day 20       600        <75                                                   Normal sera                                                                   (1)          82         <75                                                   (2)          306        <75                                                   (3)          285        <75                                                   ______________________________________                                         *Denotes exceptionally high values                                       

The assay quantitates satisfactorily between 75 and 600 μg/ml. Normalsfall in the range 0-75 μg/ml.

                  TABLE 2                                                         ______________________________________                                        Detection of influenza B antigen in immune                                    complexes                                                                     Sample       Radioactivity counts/min.                                        ______________________________________                                        Normal                                                                        (1)          1360                                                             (2)          1718                                                             First Patient                                                                 Day 1         2221*                                                           Day 4        1474                                                             Day 20       1780                                                             Second Patient                                                                Day 1         2444*                                                           Day 4        1683                                                             Day 20       1746                                                             ______________________________________                                    

It should be noted that the assay can be made to quantitatesatisfactorily from 0-75 μg/ml, but for screening purposes, when lookingfor abnormal values, this is not essential.

From these results it is clear that on Day 1--i.e. one day beforesymptoms appeared--high levels of immune complexes were present in theblood of both individuals. The level was lower by Day 4 by which timethey were recovering and by Day 20 both were normal.

The examples show that infection, even simple respiratory virusinfection could be detected by the finding of abnormal levels of immunecomplex in the blood.

Table 2 gives the results obtained with an anti-antigen (i.e.anti-influenza B virus antibody). As can be seen, it satisfactorilyidentified influenza virus in the appropriate samples.

Note that antigen was only detected in the "early" phase of infection,not during recovery; this is to be expected because after recoverybegins antigen clearance from the blood will be rapid.

EXAMPLE 3

Trypanosomiasis (sleeping sickness) in rabbits

New Zealand White rabbits were infected on 26th February with a clonepreparation of Trypanosoma brucei brucei (WIG-19). Daily blood sampleswere taken to establish whether the animals had parasites in their blood(by Leishman-stained films and microscopy), and for immune complexes bythe method described in Example 1. The weight of the animals wasrecorded each day and a careful watch was kept for signs of illness. Asummary of the results from one representative experiment is shown inthe accompanying FIG. 1.

It can be seen that after injection a progressive increase in immunecomplex levels occurred, reaching a peak between the 19th and 25thMarch. The abnormality of the animal was evident in raised immunecomplex levels prior to this time, and it was only later that weightreduction was seen. Symptoms appeared only about 30th March, andthroughout the whole experiment trypanosomes were not observed in theblood films by microscopy. Hence the immune complexes were a verysensitive indicator of the infective process which was going on in thisanimal. The complex detection was with a radiolabelled sheep anti-rabbitIgG, which favours antibody excess immune complexes.

Modifications and improvements may be made without departing from thescope of the invention.

I claim:
 1. A method of diagnosing an infected state in a biologicalfluid sample containing immune complex by determining the presence orabsence of a specific antigen in said immune complex, comprising:(a)dividing the sample into portions, (b) incubating each of the sampleportions on conglutinin to bind the immune complex to the conglutininand thereby produce respective portions of conglutinin-bound immunecomplex, (c) adding to said respective portions of the conglutinin-boundcomplex respective radiolabelled antibodies which combine specificallywith respective different antigens, (d) thereafter washing therespective portions of conglutinin-bound immune complex to which saidrespective radiolabelled antibodies have been added, and (e) detectingthe presence or absence of radioactivity in each of the respectivewashed portions, with radioactivity denoting presence of said respectivecorresponding antigen.
 2. A method according to claim 1, wherein theconglutinin is on an inert, non-organic, non-biologic solid substrate.3. A method according to claim 2, wherein the inert, non-biologic solidsubstrate is in the form of plastic balls.
 4. A method according toclaim 1, wherein prior to the addition of the radiolabelled antibodiesradiolabelled anti-immunoglobulin is added to the incubating conglutininsample mixture for reaction with said immune complex, excessradiolabelled anti-immunoglobulin is separated and the presence orabsence of radioactivity is detected in the incubatingconglutinin-sample mixture to detect the presence or absence of saidimmune complex, wherein said sample containing said radiolabelledanti-immunoglobulin and said immune complex is discarded before saidradiolabelled antibodies are added to said respective portions.
 5. Amethod according to claim 1, wherein a plurality of different labelledantibody mixtures are added one to each of the samples of saidconglutinin bound immune complex, excess of each of said mixtures isseparated, the presence or absence of said label is detected in eachsample to identify which of the mixtures has reacted with the immunecomplex, individual labelled antibodies of the mixture which reactedwith the immune complex are then added to further samples of saidconglutinin bound immune complex, excess antibody is separated in eachcase, and the presence or absence of said label is detected in eachsample to identify the antibody which has reacted with the immunecomplex.
 6. A method according to claim 1, wherein a plurality ofdifferent antibody mixtures, wherein each mixture comprises a pluralityof antibodies, each of which reacts specifically with the respectivedifferent antigen, are added, one to each of the samples of saidconglutinin bound immune complex, excess antibody mixture is separatedin each case, a polyvalent labelled reagent which reacts with thespecific antibody contained in the same antibody mixture is then addedto each of the mixtures, excess polyvalent reagent is separated in eachcase, the presence or absence of said label in each of the reactionmixtures is then detected to identify which of the mixtures has reactedwith the immune complex, individual antibodies of the mixture whichreacted with the immune complex are added to further samples of saidconglutinin bound immune complex, excess antibody is separated in eachcase, polyvalent labelled reagent known to react with the antibody isthen added to each of the reaction mixtures, excess polyvalent reagentis separated in each case, and the presence or absence of said label isdetected in each sample to identify which of the antibodies has reactedwith the immune complex.
 7. A method according to claim 1 wherein priorto addition of the radiolabelled antibodies, radiolabelledanti-immunoglobulin is added to the incubating conglutinin samplemixture for reaction with any immune complex present, excessradiolabelled anti-immunoglobulin is separated, radioactivity of boundimmunoglobulin in standard and test samples is measured and immunecomplex quantitatively determined in said test sample is based on saidstandard.
 8. A method of diagnosing an infected state in a biologicalfluid sample containing immune complex by determining presence orabsence of a specific antigen, for a particular infection, in saidimmune complex, comprising:(a) incubating the sample on conglutinin tobind the immune complex to the conglutinin, (b) adding to theconglutinin-bound immune complex enzyme-conjugated antibodies whichcombine specifically with said specific antigen; (c) washing theconglutinin-bound immune complex to which said enzyme-conjugatedantibodies have been added; (d) detecting presence or absence of saidenzyme in the washed conglutinin-bound immune complex by adding anenzyme substrate and detecting the presence or absence of a colorchange, with color change denoting presence of said enzyme andconsequent presence of said corresponding specific antigen in saidimmune complex bound to said conglutinin.
 9. A method of diagnosing aninfected state in a biological fluid sample containing immune complex bydetermining the presence or absence of a specific antigen in said immunecomplex, comprising:a. dividing the samples into portions; (b)incubating each of the sample portions on conglutinin to bind the immunecomplex to the conglutinin and thereby produce portions ofconglutinin-bound immune complex; (c) adding to said respective portionsof the conglutinin-bound immune complex respective enzyme-conjugatedantibodies which combine specifically with respective differentantigens; (d) thereafter washing the respective portions ofconglutinin-bound immune complex to which said respective enzymeconjugated antibodies have been added, and (e) detecting the presence orabsence of a respective enzyme in each of the respective washed portionsby adding an enzyme substrate and detecting the presence or absence of acolor change, with color change denoting presence of said respectiveenzyme and consequent presence of said respective corresponding antigen.10. A method according to claim 9 wherein the conglutinin is on aninert, non-organic, non-biologic solid substrate.
 11. A method accordingto claim 10 wherein the inert, non-biologic solid substrate in the formof plastic balls.
 12. A method according to claim 9 wherein prior to theaddition of the enzyme-conjugated antibodies, radiolabelledanti-immunoglobulin is added to the incubating conglutinin samplemixture for reaction with any immune complex present, excessradiolabelled anti-immunoglobulin is separated and the presence orabsence of radioactivity is detected in the incubating conglutininsample mixture to detect the presence or absence of said immune complex,wherein said sample containing said radiolabelled anti-immunoglobulinand said immune complex is discarded before said enzyme-conjugatedantibodies are added to said respective portions.
 13. A method accordingto claim 9 wherein a plurality of different enzyme-conjugated antibodieswhich react to combine specifically with different antigens are added toa plurality of samples of the conglutinin-bound complex, one to each ofthe samples of said conglutinin bound immune complex, excess of each ofsaid mixtures is separated, the presence or absence of said enzyme labelis detected in each sample to identify which of the mixtures has reactedwith the immune complex, individual enzyme labelled antibodies of themixture which reacted the immune complex are then added to furthersamples of said conglutinin bound complex, excess antibody is separatedin each case and the presence or absence of said enzyme label isdetected in each sample to identify the antibody which has reacted withthe immune complex, presence of said reacting antibody therebyidentifying the corresponding specific antigen.
 14. A method accordingto claim 9 wherein a plurality of different antibody mixtures are added,one to each of the samples of said conglutinin bound immune complex,excess antibody mixture is separated in each case, a polyvalent labelledreagent known to react with the specific antibody contained in the sameantibody mixture is then added to each of the mixtures, excesspolyvalent reagent is separated in each case, the presence or absence ofsaid label in each of the reaction mixtures is then detected to identifywhich of the mixtures has reacted with the immune complex, individualantibodies of the mixture which reacted with the immune complex areadded to further samples of said conglutinin bound immune complex,excess antibody is separated in each case, polyvalent labelled reagentknown to react with the antibody is then added to each of the reactionmixtures, excess polyvalent reagent is separated in each case, and thepresence or absence of said label is detected in each sample to identifywhich of the antibodies has reacted with the immune complex, presence ofsaid reacting antibody thereby identifying the corresponding specificantigen.
 15. A method according to claim 9 wherein prior to addition ofthe enzyme-conjugated antibodies, radiolabelled anti-immunoglobulin isadded to the incubating conglutinin sample mixture for reaction with anyimmune complex present, excess radiolabelled anti-immunoglobulin isseparated, radioactivity of bound immunoglobulin in standard and testsamples is measured and immune complex quantitatively determined in saidtest sample is based on said standard.
 16. A method of diagnosing aninfected state in a biological fluid containing immune complex bydetermining the presence or absence of an antigen forming part of saidimmune complex, comprising:(a) providing a plurality of sample portionsof said biological fluid; (b) incubating each of said sample portions onconglutinin to bind the immune complex to the conglutinin; (c) adding tosaid respective sample portions bound to the conglutinin respectivelabeled antibodies which combine specifically with respective differentantigens; (d) thereafter washing said respective sample portions boundto the conglutinin; and (e) detecting the presence or absence of a saidlabel in each of the respective washed sample portions bound to theconglutinin, with presence of said label denoting the presence of saidrespective antigen.